REVIEW ARTICLE published: 24 March 2014 doi: 10.3389/fchem.2014.00009 Allosteric modulation of oligomerization: an emerging approach to drug design

Ronen Gabizon † and Assaf Friedler*

Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem, Israel

Edited by: Many disease-related are in equilibrium between different oligomeric forms. The Youla S. Tsantrizos, McGill regulation of this equilibrium plays a central role in maintaining the activity of these University, Canada proteins in vitro and in vivo. Modulation of the oligomerization equilibrium of proteins Reviewed by: by molecules that bind preferentially to a specific oligomeric state is emerging as a Lee Fader, Boehringer Ingelheim Pharmaceuticals, Inc., USA potential therapeutic strategy that can be applied to many biological systems such Wolfgang Jahnke, Novartis as cancer and viral infections. The target proteins for such compounds are diverse Institutes for Biomedical Research, in structure and sequence, and may require different approaches for shifting their Switzerland oligomerization equilibrium. The discovery of such oligomerization-modulating compounds *Correspondence: is thus achieved based on existing structural knowledge about the specific target proteins, Assaf Friedler, Institute of Chemistry, The Hebrew University as well as on their interactions with partner proteins or with ligands. In silico design and of Jerusalem, Safra Campus, Givat combinatorial tools such as peptide arrays and phage display are also used for discovering Ram, Jerusalem 91904, Israel compounds that modulate protein oligomerization. The current review highlights some e-mail: [email protected] of the recent developments in the design of compounds aimed at modulating the †Present address: oligomerization equilibrium of proteins, including the “shiftides” approach developed in Ronen Gabizon,California Institute our lab. for Quantitative Biomedical Research (QB3), University of Keywords: protein oligomerization, peptides, HIV-1, drug design, allostery, shiftides California, Berkeley, USA

INTRODUCTION of polypeptides under thermal stress conditions. The complex Oligomerization is a common property of proteins and takes undergoes small changes in the inter-subunit interactions when place in all biological systems. It is estimated that at least 35% the temperature increases from 37 to 42–45◦C. This may enable of all proteins in cells are oligomeric (Jones and Thornton, 1996; it to distinguish normal temperatures from stress temperatures Goodsell and Olson, 2000). The properties of protein oligomers (Cabo-Bilbao et al., 2006). Protein oligomerization can also be are highly diverse: Protein oligomers may be homoligomers regulated by the binding of partner proteins (Grossman, 2001; (Wang et al., 1994; Eisenstein and Beckett, 1999; Yun et al., 2007; Fernandez-Fernandez et al., 2005; Marinho-Carvalho et al., 2006; Thulin et al., 2011) or heterooligomers (Fermi et al., 1984; Cramer De Meyts, 2008; Rajagopalan et al., 2008), metal cofactors or small et al., 2001; Unwin et al., 2002; Gomez et al., 2011) and can range molecule allosteric effectors (Kim and Raushel, 2001; Lawrence from dimers (Sapienza et al., 2007) to high order structures such et al., 2008; Selwood et al., 2008; Semenova and Chernoff, 2012). as capsids (Wu and Rossmann, 1993; Nam et al., 2011)andfib- In a notable example, Krojer et al. discovered that the bacterial rils (Craig and Woodhead, 2006; Bedrood et al., 2012). Some HtrA protease exists in an inactive hexameric state, which under- proteins form one specific active oligomeric state. This is the goes an extensive conformational change upon binding to effector case with hemoglobin, which exists in red blood cells as an α2β2 peptides. This change involves the rearrangement of the active site heterotetramer (Fermi et al., 1984), and with the acetylcholine to a catalytically active structure and induces the formation of 12- receptor that is a pentamer (Unwin et al., 2002). Other pro- mer or 24-mer oligomeric states (Krojer et al., 2010). Changes in teins are involved in dynamic oligomerization equilibria between the strength and geometry of interactions between subunits can several states with different activities, and switch between these also be modulated by ATP hydrolysis (Zhang et al., 2010). states as part of regulating their normal function. For exam- Since oligomerization is very common and is crucial for pro- ple, the enzyme carbamoyl phosphate synthase (CPS) exists in tein activity, modulating this process is a highly promising ther- equilibrium between inactive dimers and active tetramers. The apeutic strategy that can be applied to many different diseases equilibrium is regulated by allosteric inhibitors that stabilize the involving oligomeric proteins (Hayouka et al., 2007; Lawrence dimer and allosteric activators that stabilize the tetramer (Kim et al., 2008; Christ et al., 2012; Gabizon et al., 2012). Several com- and Raushel, 2001; Mora et al., 2002). In many cases, the dynamic pounds that are already in clinical use were later discovered to act assembly and disassembly of oligomers plays a central role in reg- via modulation of protein oligomerization. A well-known exam- ulating the activity of a protein, as in the case of actin (Ono, 2007), ple is that of the anti-cancer drug Taxol, which was discovered in which induces cell motility by this mechanism. the stem bark of the pacific yew during the screening of plant- Correct protein oligomerization is critical for its function derived compounds for cytotoxic activity (Wani et al., 1971). It and is therefore tightly regulated by various factors. For exam- was later discovered that Taxol binds to β-tubulin (Löwe et al., ple, the GroEL-GroES chaperonin complex assists in the folding 2001) and allosterically inhibits the assembly and disassembly

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dynamics of microtubules (Jordan et al., 1993; Derry et al., 1995), thus interfering with mitosis and inhibiting the division of cancer cells. Many compounds that modulate protein oligomerization are still being discovered indirectly by screening methods that do not target protein oligomerization. However, a growing num- ber of studies aim to discover molecules that directly target the oligomerization of a well characterized protein. The understand- ing of oligomeric protein structures and how they are regulated significantly progressed in recent years. Structures determined by NMR and X-ray crystallography enable detailed character- ization of the oligomerization interfaces and the interactions that stabilize the oligomers (see for example Fermi et al., 1984; Jeffrey et al., 1995; Lange-Savage et al., 1997; Luger et al., 1997; Walters et al., 1997; Whitson et al., 2005; Sharma et al., 2010). Methods for precise determination of the oligomeric states of a protein and their relative populations, such as size exclusion chro- matography (SEC) (Mateu and Fersht, 1998; Gotte et al., 2012; Yu et al., 2013), analytical ultracentrifugation (AUC) (Weinberg et al., 2004a; Murugan and Hung, 2012; Szymanski et al., 2013) and single molecule methods (Groulx et al., 2011; Paredes et al., FIGURE 1 | Schematic illustrations of mechanisms of modulation of 2012; Calebiro et al., 2013), improve our understanding of the protein oligomerization. (A) Direct blocking of oligomerization interfaces; thermodynamics and kinetics of oligomerization processes. Based (B) Binding to or near the oligomerization interface and stabilizing it; (C) on this knowledge, molecules that shift the oligomerization equi- Binding to several monomers simultaneously; (D) Morpheein mechanism librium of target proteins are being developed using techniques (Jaffe, 2005). The conformations determine the stoichiometry of the full ranging from de novo design to combinatorial screening. This oligomer. In this example, a ligand binds the monomer and stabilizes the conformation that promotes formation of a different oligomer. review will cover the latest developments in this field and their application to various disease-related proteins. The compounds discussed in this study are summarized in table S1. between active octamers and inactive hexamers. The transition between the two states requires dissociation into dimers followed MECHANISMS OF MODULATING THE OLIGOMERIZATION by a conformational change in the dimer and reassociation. This EQUILIBRIUM OF PROTEINS process can be modulated by allosteric effectors, such as mag- Modulation of protein oligomerization can take place by various nesium, which specifically stabilizes the octamer (Jaffe, 2005). mechanisms. In the simplest mechanism, inhibition of oligomer- Lawrence et al. used in silico screening to develop a compound ization can be achieved by molecules that bind directly to the that inhibits pea PGBS by binding specifically to the inactive hex- oligomerization interface and competitively block it (He et al., amer (Lawrence et al., 2008), thus proving the feasibility of mod- 2005), thus preventing oligomerization (Figure 1A). These com- ulating the activity of proteins by shifting their oligomerization petitive oligomerization inhibitors do not act by stabilizing a equilibrium. Many proteins exhibit characteristics indicating that specific oligomeric state and are thus outside the scope of this their oligomerization dynamics follow the morpheein mechanism review. Alternatively, molecules may stabilize a specific oligomer (reviewed in Selwood and Jaffe, 2012). by binding near the oligomerization interface (Kessl et al., 2009) (Figure 1B) or by binding to several monomers simultaneously THE TUMOR SUPPRESSOR p53 (Teufel et al., 2007)(Figure 1C). The tumor suppressor p53 is a transcription factor that is acti- A different mechanism for allosteric modulation of oligomer- vated and accumulated in the nucleus in response to oncogenic ization equilibria was described by Jaffe et al. (Jaffe, 2005). In this stress. Following its induction, p53 binds specific promoters in mechanism, a lower oligomeric form of the protein can exist in the genome and activates the transcription of a wide array of different conformations (called morpheeins), and each confor- target genes, aimed at eliminating the threat of malignant trans- mation dictates a defined stoichiometry for the higher oligomer. formation (Levine, 1997; Vogelstein et al., 2000; Ryan et al., 2001; Transition between the oligomeric states requires dissociation of Michael and Oren, 2002). p53 is mutated in over 50% of all can- theoligomerandachangeinconformationbeforetheother cer cases, highlighting the vital role it plays in tumor suppression. oligomeric state is formed. Therefore, molecules that allosterically The majority of cancer-associated mutations in p53 occur in its stabilize a certain conformation of the lower oligomer will shift DNA binding core domain (Levine, 1997). the oligomerization equilibrium toward the corresponding higher p53isactiveasahomotetramer(Chene, 2001)andits oligomer (Figure 1D). This mechanism has been shown for the tetramerization is mediated by a structurally independent enzyme phorphobilinogen synthase (PBGS), which is involved in tetramerization domain (p53Tet, residues 326–355) (Clore et al., tetrapyrrole metabolism and plays a crucial role in cellular res- 1994; Lee et al., 1994; Jeffrey et al., 1995). Tetramerization of p53 piration (Jaffe and Lawrence, 2012). PBGS exists in equilibrium is vital to its function and plays a central role in the regulation

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of p53 activity. p53 tetramers bind p53 DNA response elements that increasing the flexibility of the ligand increases its affinity to more tightly than dimers and monomers, and only tetramers can the R337H by enabling it to accommodate the optimal geometry induce transcription of p53 target genes (Weinberg et al., 2004b; for binding more easily (Gordo et al., 2011). However, the activity Menendez et al., 2009). Tetramerization also affects the cellular of this ligand was tested in water, and experiments at physiologi- localization of p53: the Nuclear Export Signal (NES) of p53 is cal ionic strength did not show any activity (Kamada et al., 2010). located within the tetramerization domain and is shielded in p53 Based on this work, Kamada et al. Designed larger ligands with a tetramers, preventing nuclear export of p53 tetramers (Stommel calix[6]arene group with different positively charged end groups. et al., 1999). However, in monomers and dimers of p53 the NES One of the ligands, containing imidazole groups, increased the isexposedandp53isthusexportedfromthenucleustothe thermal stability of the R337H and enhanced the transcriptional cytoplasm, where it is degraded via the ubiquitin-proteasome activity of p53 R337H in cells (Kamada et al., 2010). pathway. A different approach to discovering molecules that stabilize The oligomerization equilibrium of p53 is regulated by inter- p53 tetramers was used in our laboratory (Gabizon et al., 2008, actions with other proteins, such as proteins from the 14-3- 2012). We used the natural protein-protein interactions of the p53 3 and S100 families (Fernandez-Fernandez et al., 2005, 2008; tetramerization domain or nearby regions for developing pep- Rajagopalan et al., 2008; Słomnicki et al., 2009; Van Dieck et al., tides that bind p53 in or near its tetramerization domain and 2009a) and numerous kinases (Delphin et al., 1997; Gotz et al., may thus stabilize the tetramer. The interaction between p53Tet 1999). Post translational modifications also have an effect on and the HIV-1 Tat protein (Longo et al., 1995) was character- p53 oligomerization, either by directly affecting tetramer stabil- ized using peptide mapping. Two peptides from HIV-1 Tat bound ity (Nomura et al., 2009; Yakovlev et al., 2010) or by modulating p53Tet, and the interaction of a peptide derived from the arginine theinteractionsofp53withotherproteins(Rajagopalan et al., rich motif of Tat with p53Tet was characterized. The Tat-derived 2008; Van Dieck et al., 2009b). Recently, using fluorescence corre- peptide bound all oligomeric forms of p53Tet without preference lation spectroscopy in single cells, Gaglia et al. showed that DNA and thus was not a potential candidate for modulating p53Tet damage causes the shifting of the oligomerization equilibrium of oligomerization (Gabizon et al., 2008). In a following work, we p53 toward tetramers, and that this change is sufficient to acti- used a combinatorial approach and designed a peptide array vate the transcription of p53 target genes even without the net derived from proteins known to bind the C terminal domain accumulation of p53 (Gaglia et al., 2013). The importance of (CTD) of p53 (p53CTD, residues 293–393). Screening the array tetramerization for p53 function makes p53 an attractive thera- with recombinant p53CTD resulted in the identification of 10 peutic target for compounds that modulate protein oligomeriza- peptides that bound p53CTD. Several of these peptides increased tion. Several recent projects utilized different strategies to shift the the thermal stability of the p53CTD and bound specifically to p53 oligomerization equilibrium of p53 toward the active tetramer. tetramers in AUC experiments (Gabizon et al., 2012)(Figure 2C). Ligands containing several spaced cationic groups bound the Other cancer related proteins may also be potential targets p53 tetramerization domain and stabilized p53 tetramers. These for molecules that modulate their oligomerization. Gray et al. ligands were developed using a combination of intuitive design developed a fluorescent monoclonal antibody assay for deter- with computational and combinatorial methods. Salvatella et al. mining the extent of oligomerization of the oncoprotein AGR2 designed a tetraguanidinium ligand that binds to a patch of (Gray et al., 2013). They found that a peptide derived from negatively charged residues on the surface of the p53 tetramer- the disordered N-terminal domain (NTD) of AGR2 stabilizes ization domain (Figure 2A), facing outwards from the dimer- AGR2 oligomers, and that oligomerization of AGR2 enhances dimer interface (Salvatella et al., 2004). This ligand was used by its binding to its chief partner protein reptin. This highlights Martinell et al. as a basis for the computational design of a peptide the therapeutic potential of compounds that modulate AGR2 with four arginine residues with similar spacing as the guani- oligomerization. dinium groups in the original ligand. The new peptide (CAN4) Mdm2 and mdmX are negative regulators of p53 that medi- bound p53Tet with a Kd of 8 μM and increased the thermal stabil- ate polyubiquitination and degradation of p53. Graves et al. ity of p53Tet by 2◦C. The same group later synthesized a library of developed compounds that bind mdm2 and mdmX and induce modified peptides and tested their binding to p53Tet. Several pep- dimerization of the proteins. The p53-binding interface is buried tides in the library bound p53Tet with affinities as low as 0.8 μM in the dimerization interface, thus inhibiting the binding of p53 (Martinell et al., 2006). by mdm2 and mdmX (Figure 3A). The compounds activate the Gordo et al. focused on the cancer associated R337H mutant p53 transcriptional pathway in cells and induce apoptosis of of p53, in which a critical hydrogen bond is lost due to the cancer cells (Graves et al., 2012). mutation, resulting in destabilization of the p53 tetramer. They Another example of anti-cancer compounds that act by designed a calix[4]arene with 4 guanidiniomethyl groups, in promoting protein oligomerization is given by the work of which a hydrophobic calixarene group binds to the hydropho- Anastasiou et al. (2012). In cancer cells, inhibition of the enzyme bic pocket formed in the dimer-dimer interface of the p53Tet M2 (PKM2) by phosphotyrosine-containing pro- tetramer and the guanidinium groups form hydrogen bonds with teins increases the availability of glycolytic metabolites for the acidic residues located on different monomers (Figure 2B). The support of cell proliferation. The authors characterized two small designed ligand bound the R337H mutant and increased the ther- molecule activators of PKM2. The compounds activate PKM2 mal stability of the mutant to the same level as the wild type by stabilizing its tetrameric state, prevent inhibition of PKM2 p53Tet (Gordo et al., 2008). In a later work, Gordo et al. showed by phosphotyrosine-containing protein, alter the metabolism of

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FIGURE 2 | Modulation of p53 oligomerization. (A) Left: Structure of the and imidazole calix[6]arene described in Kamada et al. (2010) (2). Right: tetraguanidinium ligand described in Salvatella et al. (2004) and sequence structure of the acidic patch targeted by these molecules; (C) Discovery of of the peptide CAN4 described in Martinell et al. (2006). Right: Stick peptides that bind specifically to tetrameric p53. A peptide array derived model and space filling model (indicated residues in red) of the acidic from p53-binding proteins was screened for binding to p53CTD (left). The patch on the surface of the p53 tetramerization domain targeted by the binding of the identified peptides was quantified by fluorescence two molecules. Structures from pdb 1PET (Lee et al., 1994); (B) Left: anisotropy (center) and their effect on p53 oligomerization was Structure of guanidinium-calix[4]arene described in Gordo et al. (2008) (1) characterized using AUC (right). cancer cells and inhibit cancer cell proliferation. Structural stud- processing of viral polyproteins or the maturational processing ies of the compounds with PKM2 revealed that the compounds of precapsids. These proteases are frequently oligomeric, as in bind at the interface between two monomers and stabilize the HIV (Lange-Savage et al., 1997) and hepatitis C virus (Li et al., tetrameric structure (Figure 3B). 2010). The function of viral capsid proteins is derived from Another potential target may be BAK, a mitochondrial mem- their oligomerization properties, and the high order structures of brane protein involved in apoptosis. Following cell stress, p53 numerous capsids have been characterized, as in the case of canine binds to BAK and induces BAK oligomerization (Leu et al., 2004; parvovirus (Wu and Rossmann, 1993), Hepatitis B virus (HBV) Pietsch et al., 2008), a critical stage in mitochondrial apoptosis. (Katen et al., 2013), and HIV (Zhao et al., 2013). Other promi- Thus molecules that bind and induce oligomerization of BAK nent viral proteins that are active as oligomers include may have potential as anti-cancer drug leads. (Cherepanov et al., 2003) and reverse transcriptases (Smerdon et al., 1994). Targeting the oligomerization of these proteins is VIRAL PROTEINS emerging as a promising therapeutic strategy. Oligomeric proteins play vital roles in the replication cycles Considerable work has been performed on directly inhibit- of viruses. Many viruses encode proteases that catalyze the ing the oligomerization of viral proteins using molecules that

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FIGURE 3 | (A) Crystal structure of mdmX with the inhibitor RO-2443. A 2012); (B) Crystal structure of pyruvate kinase M2 (PKM2) with the activator dimer of RO-2443 (sticks, yellow, and cyan) induces dimerization of mdmX DASA-58. The four monomers are in red, green, blue and yellow and are (each monomer in different shade of gray). The binding sites for p53 (red and semitransparent. DASA-58 is in cyan. The second activator TEPP-46 binds at magenta) are blocked in the dimer. Structure from pdb 3U15 (Graves et al., the same site. Structure from pdb 3ME3 (Anastasiou et al., 2012). bind to their oligomerization interfaces. Peptides derived from complex (Engelman et al., 1991). This step is performed by the oligomerization interfaces can inhibit the oligomerization and tetrameric IN (Li and Craigie, 2005; Li et al., 2006)inthenucleus activity of such proteins, as was shown for the HIV-1 reverse tran- with the assistance of cellular proteins, especially LEDGF/p75, scriptase (Divita et al., 1994; Morris et al., 1999; Depollier et al., which promotes IN tetramerization and tethers it to the chromo- 2005)andintegrase(Sourgen et al., 1996; Maroun et al., 2001; somes (Cherepanov et al., 2003; Maertens et al., 2003; Emiliani Zhao et al., 2003). Non peptidic inhibitors of protein oligomeriza- et al., 2005). The protein-protein interactions of IN are emerg- tion have also been characterized (Rodríguez-Barrios et al., 2001; ing as promising therapeutic targets (Maes et al., 2012), in Bonache et al., 2005; Koh et al., 2007; Vidu et al., 2010; Tintori particular the IN-LEDGF/p75 interaction (Christ and Debyser, et al., 2012). This subject has been reviewed elsewhere and is 2013). outside the scope of the current review (Camarasa et al., 2006). The importance of the integration step within the HIV repli- Inhibition of viral proteins can also be achieved by ligands cation cycle and the lack of mammalian homologs for IN both that do not bind the oligomerization interface, but rather mod- make IN an attractive therapeutic target. However, only two IN ulate the oligomerization of the protein allosterically. One of the inhibitors (Raltegravir and Elvitegravir) are currently used in the main targets for this approach is the HIV-1 protein clinic as anti-HIV drugs (Serrao et al., 2009; Messiaen et al., (IN), which catalyzes the integration of the viral cDNA into the 2013). The rapid development of resistant strains (Mouscadet host genome (Delelis et al., 2008), a crucial step in the HIV-1 et al., 2010) emphasize the need to develop new drugs that func- replication cycle (Sherman and Greene, 2002). Integration pro- tion by different mechanisms, and efforts are made by numerous ceeds by two steps, each performed by a specific IN oligomer: laboratories to discover novel IN inhibitors. In most cases, the (i) 3 end processing, in which IN removes a GT dinucleotide modulation of IN oligomerization was not the intended result from the 3 termini of the long terminal repeats (LTRs) in the and candidate compounds were screened for inhibition of IN cat- viralDNA.ThisstepisperformedbyINdimers(Deprez et al., alytic activity or inhibition of IN-LEDGF/p75 binding. However, 2001; Guiot et al., 2006) bound to each LTR in the cytoplasm; many of the most promising IN inhibitors discovered recently (ii) strand transfer, in which the viral DNA is integrated into were later shown to act primarily by shifting the oligomerization the host DNA following nuclear import of the stable synaptic equilibrium of IN.

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Kessl et al. studied IN inhibitors derived from chicoric acid, monomers, thus stimulating oligomerization (Figure 4A). The which inhibit the strand transfer activity of IN at micromolar authors postulate that the compound induces the formation of concentrations (Kessl et al., 2009). They developed a method conformationally rigid IN tetramers that are unable to bind DNA to measure the rate of subunit exchange between IN oligomers. in the necessary orientation for catalysis. The method uses His6-labeled IN and non-labeled IN, which are In our laboratory we discovered peptides that bind IN and allowed to equilibrate separately and then mixed. The exchange shift its oligomerization equilibrium toward the tetramer, thus +2  of subunits is monitored by pulling down the His6-IN with Ni inhibiting the 3 -end processing catalytic activity and preventing beads and measuring the amount of non-labeled IN pulled down viral replication. We termed these peptides “Shiftides.” The first by SDS PAGE. One of the compounds significantly reduced the anti-IN shiftides were derived from the IN-binding site in the extent of subunit exchange, indicating that the oligomers were cellular protein LEDGF/p75 (Hayouka et al., 2007)(Figure 4B). stabilized and thus dissociated more slowly. Molecular docking These peptides bound IN, shifted its oligomerization equilibrium and mutational analysis indicated that the compound binds at a toward the tetramer and inhibited IN activity in vitro and in cleft located at the interface between two monomers of the IN HIV-1 infected cells (Hayouka et al., 2010a). We then synthesized catalytic core domain (CCD), while forming contacts with both a series of cyclic peptides derived from the IN binding sites in

FIGURE 4 | Inhibiting of HIV-1 IN by modulating its oligomerization (magenta). The sequences of peptide inhibitors derived from LEDGF equilibrium. (A) Structure of the IN inhibitor described by Kessl et al. (Hayouka et al., 2007), Rev (Hayouka et al., 2008) and from combinatorial (2009) and Possible binding sites. The two monomers of the IN CCD are screening (Maes et al., 2009) are given. Structure from pdb 2B4J in two shades of gray. The possible binding sites for the inhibitor are in (Cherepanov et al., 2005); (C) Structures of IN inhibitors described in the orange and magenta. Structure from pdb 1EXQ (Chen et al., 2000). (B) review (from left to right): c(MZ 4-1), a cyclic derivative of LEDGF 361–370 Structure of the complex between the IN CCD (dimer, two shades of gray) (Hayouka et al., 2010b). Amino acid residues are shown in red; Inhibitors and the IN binding domain of LEDGF/p75 (green). The LEDGF sequences CX05168, CX05045, and CX014442 Described by Christ et al. (2010, 2012); that mediate IN binding are residues 361–370 (red) and 402–411 inhibitor BI 224436 (Fader et al., in press).

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LEDGF/75. One of the peptides inhibited IN as much as the lin- and Panja, 2013)andyetbeabletodissociateuponcellentryand ear peptide while being more stable in cells (Figure 4C)(Hayouka release the viral DNA and proteins into the host cell. Incorrect et al., 2010b). The mechanism of binding of the cyclic peptides to formation of the viral capsid can be detrimental to the replication IN depended on their ring size—while peptides with smaller rings of viruses and thus mutations that destabilize or alter the struc- bound preferably to IN dimers and stabilized them, larger rings ture of the capsid highly reduce the infectivity of viruses, as in the promoted binding and stabilization of IN tetramers (Hayouka case of HIV (Noviello et al., 2011)andHBV(Tan et al., 2013). et al., 2010b). A similar mechanism of inhibition was discov- Therefore, many inhibitors targeted against capsid proteins are ered for peptides derived from the viral protein Rev (Figure 4B) currently being developed. (Hayouka et al., 2008), which binds and inhibits IN (Rosenbluh The HIV-1 capsid protein (CA) is one of the main targets for et al., 2007). IN-inhibitory shiftides were also developed by a inhibition. CA is composed of two helical domains, the NTD combinatorial approach: a 20 residue peptide termed IN1, which and the CTD, which are connected via a flexible linker. The basic was identified by a yeast two hybrid assay, bound to tetrameric IN, structural unit of the capsid is a hexameric ring formed by NTD- shifted the oligomerization equilibrium of IN toward the tetramer NTD interactions, and reinforced by intermolecular NTD-CTD and inhibited IN activity (Figure 4B)(Armon-Omer et al., 2008; interactions (Figure 5A). Dimeric CTD-CTD interactions link Maes et al., 2009). In comparison, peptides comprising residues between the hexameric rings, and the full capsid has a fullerene- 1–10 or residues 11–20 of IN1 bound to IN dimers, indicating like structure (Pornillos et al., 2009). Correct assembly of the that IN1 may stabilize tetrameric IN by bridging two IN dimers capsid may be very sensitive to small changes in the strength (Maes et al., 2009). and geometry of the inter-subunit interactions. Unlike in the A major class of novel IN inhibitors are termed Allosteric case of HIV-1 integrase, most HIV inhibitors that target CA were IN Inhibitors (ALLINI’s) (Tsantrizos et al., 2007; Christ et al., intended for inhibition of capsid formation and were discovered 2012; Tsiang et al., 2012; Engelman et al., 2013; Fader et al., by rational structure-based approaches or screenings that used in press). Christ et al. used a combination of computational and in vitro capsid assembly assays. experimental methods to discover IN-inhibiting compounds. The Tang et al. used virtual screening to search for compounds authors initially aimed to find inhibitors for the IN-LEDGF/p75 that bind and inhibit CA. One of the compounds discovered, interaction and used virtual screening to design a library of com- CAP-1, inhibits HIV-1 infectivity by specifically interfering with pounds that may bind to the LEDGF/p75 binding sites in IN the formation of viral capsids (Tang et al., 2003). CAP-1 inhib- and inhibit the IN-LEDGF/p75 interaction. Several compounds ited capsid formation in vitro and caused the formation of viral with micromolar activities were discovered (Christ et al., 2010). capsids with abnormal morphologies. 1H-15N HSQC measure- Further optimization yielded the compound CX14442, which ments showed that CAP-1 binds the NTD at the apex of the inhibited the IN-LEDGF/p75 interaction at submicromolar con- helical bundle, and inhibits the NTD-CTD interaction necessary centrations (Figure 4C)(Christ et al., 2012). The authors discov- for hexamer stabilization. Kelly et al. further characterized the ered that beyond inhibiting the IN-LEDGF/p75 interaction, the binding of CAP1 to CA using X-ray crystallography and NMR, compounds enhance oligomerization of IN and directly inhibit and observed that CAP1 binds into a hydrophobic pocket formed IN activity. Kessl et al. further studied the mechanism of action of by the displacement of Phe32 (Kelly et al., 2007)(Figure 5B). these compounds as well as a similar IN inhibitor discovered by Other compounds that inhibit CA by a mechanism simi- high throughput screening for 3 processing activity (Figure 4C) lar to CAP1 have been studied. Lemke et al. developed a high (Tsantrizos et al., 2007). The ALLINI’s bound at the dimerization throughput assay for testing the effect of compounds on cap- interface between two CCD monomers, promoted IN multimer- sid formation and used it to screen a large library for CA- ization and increased the thermal stability of IN. Studies of an IN inhibitors. This led to the discovery of new inhibitors derived mutant which is resistant to inhibition by ALLINI’s showed that from Benzimidazole (BM) and benzodiazepine (BD) (Lemke inhibition of IN activity is achieved mainly by the promotion of et al., 2012)(Figure 5B). While both families bound CA at the IN multimerization and not by inhibition of the IN-LEDGF/p75 same site (which is identical to the CAP-binding site), their interaction (Feng et al., 2013). Furthermore, Jurado et al. found modes of binding were different and had distinct effects on that ALLINI’s promote IN multimerization in virions, and that HIV-1 replication: while BD compounds inhibited capsid for- virions produced in the presence of ALLINI’s are not infectious mation and release, BM compounds inhibited the formation of and have no reverse transcriptase or integrase activity (Jurado the mature, conical capsid after release from the cell. In later et al., 2013). One of the compounds, BI 224436, has an EC50 studies, the compounds were modified and improved - Tremblay of 11–27 nM and has recently entered clinical trials (Fader et al., et al. systematically modified the functional groups in a BM in press). These studies highlight the versatility and potency of scaffold, yielding CA-inhibitors with IC50 values below 0.1 μM compounds that inhibit IN by modulating its oligomerization (Tremblay et al., 2012). Goudreau et al. developed BD-based equilibrium. compounds with IC50 values below 1 μM(Goudreau et al., Viral capsid proteins are also a promising target for com- 2013). pounds that modulate oligomerization. Viral capsids are large, Recently, Goudreau et al. characterized a novel family of BM- highly symmetric oligomers comprised of one or several types of based CA inhibitors that bind the NTD in a distinct site from monomers, and may contain hundreds of subunits (Mateu, 2013). CAP-1(Goudreau et al., 2013). One of the compounds induces the The function of capsid proteins requires that they form highly sta- formation of an NTD-dimer with a non-native geometry (Lemke ble capsids that can withstand high internal pressures (Molineux et al., 2013)(Figure 5C).

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FIGURE 5 | Molecules that target capsid assembly. (A) Structure of HIV-1 the bottom. (C) Formula of CA-binding compound described by Lemke et al. capsid protein hexamer. Different monomers are in Green, red, blue, (2013). (D) Formula of the CA-binding compounds PF74 (described by Shi magenta, cyan, and orange. The NTD and CTD of every monomer are in et al., 2011) and BI-2 (described by Lamorte et al., 2013) (E) Structure of HIV-1 different shades of the same color. Structure from pdb 1VUU (Zhao et al., capsid protein CTD in the absence (left) and presence (right) of the capsid 2013). (B) Structure of HIV-1 capsid protein NTD in the absence (left) and assembly inhibitor (CAI). CAI is in orange. The helix from which the peptide presence (right) of CAP-1. Phe32 is shown in yellow, CAP-1 is shown in CAC1 was derived (Bocanegra et al., 2011) is in cyan. The sequence of CAI magenta. Structures from pdb files 1VUU (Zhao et al., 2013) and 2JPR (Kelly and the structure of the optimized peptide NYAD-1 (Zhang et al., 2008)are et al., 2007). The formulas of CAP-1 and a representative Benzimidazole given below. Structures from pdb files 1AUM (Gamble et al., 1997) and 2BUO (Goudreau et al., 2013) and Benzodiazepine (Tremblay et al., 2012) are given at (Ternois et al., 2005).

Several compounds that target the CA NTD were discovered et al. discovered pyrrolopyrazolone based HIV inhibitors that by screening of compound libraries for inhibition of HIV repli- bind CA at a similar binding site to PF74 and increase the sta- cation in cells, with further studies revealing that CA was the bility of the viral capsid, thus interfering with the nuclear import target. Blair et al. and Shi et al. discovered the compound PF- of the stable synaptic complex (Lamorte et al., 2013)(Figure 5D). 74, which inhibits HIV replication in cells by binding to the These results indicate that the viral replication cycle can be very NTD of CA and destabilizing the capsid structure (Blair et al., sensitive to small changes in capsid stability in either direction, 2010; Shi et al., 2011). This causes premature uncoating of the which further highlights the therapeutic potential of CA-binding virionduringthereplicationcycle.Ontheotherhand,Lamorte molecules.

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The CTD of CA is also a target for inhibition. Sticht et al. used phage display screening to identify peptides that bind CA and inhibit capsid formation. The authors discovered a peptide, termed capsid assembly inhibitor (CAI), that binds to the CTD and inhibits the formation of the mature capsid (Sticht et al., 2005). X-ray crystallography of the peptide-bound CTD (Ternois et al., 2005) showed that the peptide adopts a helical conforma- tion and binds into a hydrophobic groove in the CTD, forming a five helix bundle. The binding of the peptide significantly alters the geometry of the dimerization interface (Figure 5E). Therefore, although the peptide does not directly bind the dimer- ization interface and does not destabilize the CTD dimer, it alters the geometry of the dimer interface, thus preventing the forma- tion of the mature capsid. In a later study, the sequence of CAI was optimized using hydrocarbon stapling in order to stabilize the helical secondary structure of the peptide. This resulted in the peptide NYAD-1, which had improved cell penetration and activity in vivo (Zhang et al., 2008). Bocanegra et al. used a rational design approach to inhibit cap- sid assembly. The authors synthesized a peptide, termed CAC1, derived from a helix in the dimerization interface of the CTD (Figure 5E), and also made several modifications in the peptide to increase its solubility and its affinity to the CTD. The peptides bound directly to the dimerization interface of CTD, inhibited capsid assembly and also had antiviral activity in cells (Bocanegra FIGURE 6 | (A) Structure of the enterovirus 71 capsid proteins with WIN et al., 2011). 51711.Three subunits of the capsid are in blue, green and red. WIN 51711 is in Capsid-binding molecules have been studied for other viruses. yellow. Based on Plevka et al. (2013); (B) Formulas of HBV capsid binding inhibitors (Stray et al., 2005; Stray and Zlotnick, 2006). Plevka et al. used X-ray crystallography to study the effect of the capsid binding inhibitor WIN 51711 on the replication of enterovirus 71, which is associated with foot and mouth disease Alzheimer’s disease (Gilbert, 2013), Parkinson’s disease and (Plevka et al., 2013). They discovered that WIN 51711 binds a diseases (Salvatella, 2013). The inhibition of fibrillation is pocket in one of the subunits involved in capsid formation and amajorgoalindrugdevelopment.Amyloidβ fibrillation can be increases the stability of the capsid, thus restricting the dynam- inhibited by short peptides derived directly from the sequences ics of the capsid necessary for genome release and lowering the that mediate fibrillation (Tjernberg et al., 1996). Others meth- infectivity of the virus (Figure 6A). ods involve rational design of inhibitors based on the structure of The HBV is also being targeted by capsid-binding inhibitors. the interface between monomers (Sato et al., 2006; Sievers et al., Heteroaryldihydropyrimidines (HAPs) are a class of compounds 2011). This subject has been further reviewed elsewhere (Soto and that inhibit HBV replication in tissue culture by interfering with Estrada, 2005; Belluti et al., 2013). capsid formation (Deres et al., 2003). The HBV capsid is made The monomer-fibril equilibrium can also be shifted toward the of 120 dimers of capsid protein (Cp) arranged in icosahedral fibril, as has been shown in our laboratory for non-muscle myosin symmetry (Bottcher et al., 1997). Stray et al. studied the effects type II (NMII). NMII undergoes dynamic filament assembly of the two HAP compounds, HAP-1 and BAY 41-4109, on cap- and participates in cellular processes such as cell migration and sid formation in vitro (Stray et al., 2005; Stray and Zlotnick, cytokinesis. Ronen and Rosenberg et al. investigated the role of 2006)(Figure 6B). While at substoichiometric concentrations the non-helical C-terminal tailpiece in filament assembly (Ronen the compounds increase the rate of capsid formation, at high et al., 2010). They found that the tailpiece is intrinsically disor- concentrations they misdirect capsid formation and induce the dered and is divided into two oppositely charged regions. The formation of aberrant structures. The mechanism by which the positively charged part of the tailpiece interacts with an assem- compounds inhibit viral replication is still unclear—it is possible bly incompetent fragment of NMII and induces its filamentation, that the compounds do not antagonize HBV by directly inhibiting while the negatively charged part affects the morphology of the capsid formation, but rather disrupt the coordination of capsid filaments. assembly with other stages of the replication, or inhibit struc- tural transitions necessary for the formation of mature, infectious CONCLUSION capsids. Oligomerization plays a crucial role in the activity of many disease-related proteins and is therefore a promising target for FIBRIL-FORMING PROTEINS therapeutic intervention. The molecules presented here were The equilibrium between monomers and fibrils plays a major role discovered by methods ranging from combinatorial screening in many diseases, particularly neurodegenerative diseases such as methods such as phage display to rational, structure-based design.

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optimization of shiftide activity as well as improving pharma- cological properties such as cell permeability (Wang et al., 2014) and proteolytic stability (Moretto et al., 2006; Dong et al., 2012) (Figure 7). Therefore, the shiftide approach can be applied effec- tively to a wide range of disease related proteins with dynamic oligomerizaion equilibria. The number of disease-related oligomeric proteins that are being studied and characterized continues to grow (Lawrence et al., 2008; Ferré et al., 2010). A prominent example is G-protein coupled receptors (GPCRs). Many GPCRs are now known to form homo-and hetero-oligomers (Ferré et al., 2010), and the oligomerization of GPCRs can be critical to their activity (Jones et al., 1998) or significantly alter it (Azdad et al., 2008). The binding of agonists and antagonists can also induce conforma- tional changes in the dimerization interface of GPCRs (Guo et al., 2005). Therefore, allosteric modulation of oligomerization may be a powerful approach for the development of drugs that target GPCRs in the future. As the number of potential targets grows, we expect that modulation of protein oligomerization will become a central therapeutic strategy for a variety of diseases.

ACKNOWLEDGMENTS Assaf Friedler was supported by a starting grant from the European Research Council under the European Community’s FIGURE 7 | Shiftides—peptides that modulate the oligomerization Seventh Framework Programme (FP7/2007-2013)/ERC Grant equilibrium of proteins. Shiftides can be developed using combinatorial ◦ screening or rational design, and can be modified easily to improve their agreement n 203413 and by the Minerva Center for Bio- activity and pharmacological properties. An example of a protein in a hybrid complex systems. Ronen Gabizon was supported by the dimer—tetramer equilibrium is given, but the principle can be applied to any Adams Fellowship from the Israel Academy of Science and the oligomerization equilibrium. Humanities.

SUPPLEMENTARY MATERIAL In most cases a combination of several methods was used. Many The Supplementary Material for this article can be found of the compounds were discovered in experiments that did not online at: http://www.frontiersin.org/journal/10.3389/fchem. aim for modulating protein oligomerization, and their mecha- 2014.00009/abstract nism of action was elucidated later. However, as the knowledge of the oligomerization states and structure of the target proteins accumulates and methods that measure changes in the stability or REFERENCES structure of the oligomers become widespread, a growing num- Anastasiou, D., Yu, Y., Israelsen, W. J., Jiang, J., Boxer, M. B., Hong, B. S., et al. (2012). Pyruvate kinase M2 activators promote tetramer formation and ber of active compounds are designed and screened for allosteric suppress tumorigenesis. Nat. Chem. Biol. 8, 1008. doi: 10.1038/nchembio1212- modulation of protein oligomerization. It is therefore not surpris- 1008b ing that most of the compounds being developed target proteins Armon-Omer, A., Levin, A., Hayouka, Z., Butz, K., Hoppe-Seyler, F., Loya, S., et al. with well characterized structures and oligomerization equilibria. (2008). Correlation between shiftide activity and HIV-1 integrase inhibition by a peptide selected from a combinatorial library. J. Mol. Biol. 376, 971–982. doi: 10.1016/j.jmb.2007.11.095 THE SHIFTIDES CONCEPT Azdad, K., Gall, D., Woods, A. S., Ledent, C., Ferre, S., and Schiffmann, In our laboratory we focus on peptides as tools for modulat- S. N. (2008). Dopamine D2 and Adenosine A2A Receptors Regulate ing protein oligomerization and we term them “shiftides.” These NMDA-Mediated Excitation in Accumbens Neurons Through A2A-D2 peptides bind specifically to a particular oligomeric state of the Receptor Heteromerization. Neuropsychopharmacology 34, 972–986. doi: 10.1038/npp.2008.144 target protein and stabilize it. By doing so, these peptides shift Bedrood, S., Li, Y., Isas, J. M., Hegde, B. G., Baxa, U., Haworth, I. S., et al. the oligomerizaton equilibrium toward this specific oligomeric (2012). Fibril structure of human islet amyloid polypeptide. J. Biol. Chem. 287, state. This way it is possible either to activate a protein by 5235–5241. doi: 10.1074/jbc.M111.327817 stabilizing an active oligomer or inhibit a protein by stabiliz- Belluti, F., Rampa, A., Gobbi, S., and Bisi, A. (2013). Small-molecule β ing an inactive oligomer. In this review, we have demonstrated inhibitors/modulators of amyloid- peptide aggregation and toxicity for the treatment of Alzheimer’s disease: a patent review (2010 – 2012). Expert Opin. the development of shiftides that target several proteins such Ther. Pat. 23, 581–596. doi: 10.1517/13543776.2013.772983 as HIV-1 integrase (Hayouka et al., 2007; Maes et al., 2009), Blair, W. S., Pickford, C., Irving, S. L., Brown, D. G., Anderson, M., Bazin, R., p53 (Gabizon et al., 2012) and non-muscle myosin IIC (Ronen et al. (2010). HIV Capsid is a Tractable Target for Small Molecule Therapeutic et al., 2010). Shiftides can be discovered using rational design Intervention. PLoS Pathog. 6:e1001220. doi: 10.1371/journal.ppat.1001220 Bocanegra, R., Nevot, M., Doménech, R., López, I., Abián, O., Rodríguez-Huete, based on the sequences of proteins known to bind the target A., et al. (2011). Rationally designed interfacial peptides are efficient in vitro protein, using combinatorial approaches, or combining the two inhibitors of HIV-1 capsid assembly with antiviral activity. PLoS ONE 6:e23877. methods. The versatile chemistry of peptides enables the facile doi: 10.1371/journal.pone.0023877

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Frontiers in Chemistry | Chemical Biology March2014|Volume2|Article9| 14 Gabizon and Friedler Allosteric modulation of protein oligomerization

Zhang, H., Zhao, Q., Bhattacharya, S., Waheed, A. A., Tong, X., Hong, A., et al. relationships that could be construed as a potential conflict of (2008). A cell-penetrating helical peptide as a potential HIV-1 inhibitor. J. Mol. interest. Biol. 378, 565–580. doi: 10.1016/j.jmb.2008.02.066 Zhang, J., Baker, M. L., Schroder, G. F., Douglas, N. R., Reissmann, S., Jakana, J., Received: 26 December 2013; accepted: 22 February 2014; published online: 24 March et al. (2010). Mechanism of folding chamber closure in a group II chaperonin. 2014. Nature 463, 379–383. doi: 10.1038/nature08701 Citation: Gabizon R and Friedler A (2014) Allosteric modulation of protein oligomer- Zhao,G.,Perilla,J.R.,Yufenyuy,E.L.,Meng,X.,Chen,B.,Ning,J., ization: an emerging approach to drug design. Front. Chem. 2:9. doi: 10.3389/fchem. et al. (2013). Mature HIV-1 capsid structure by cryo-electron microscopy 2014.00009 and all-atom molecular dynamics. Nature 497, 643–646. doi: 10.1038/ This article was submitted to Chemical Biology, a section of the journal Frontiers in nature12162 Chemistry. Zhao, L., O’Reilly, M. K., Shultz, M. D., and Chmielewski, J. (2003). Interfacial Copyright © 2014 Gabizon and Friedler. This is an open-access article distributed peptide inhibitors of HIV-1 integrase activity and dimerization. Bioorg. Med. under the terms of the Creative Commons Attribution License (CC BY). The use, dis- Chem. Lett. 13, 1175–1177. doi: 10.1016/S0960-894X(03)00040-4 tribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in Conflict of Interest Statement: The authors declare that the research accordance with accepted academic practice. No use, distribution or reproduction is was conducted in the absence of any commercial or financial permitted which does not comply with these terms.

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